U.S. patent application number 10/470986 was filed with the patent office on 2004-06-17 for method for genotype determination.
Invention is credited to Costa, Jean-Marc.
Application Number | 20040115684 10/470986 |
Document ID | / |
Family ID | 8176348 |
Filed Date | 2004-06-17 |
United States Patent
Application |
20040115684 |
Kind Code |
A1 |
Costa, Jean-Marc |
June 17, 2004 |
Method for genotype determination
Abstract
The present invention is directed to a new method for genotype
determination at a specific gene locus of an individual or a fetus
comprising (i) amplifying a first sequence of said gene locus and a
second sequence of a second reference gene locus from DNA
originating from a sample containing biological material of said
individual or fetus (ii) Monitoring both amplifications preferably
in real time and determining the amount of amplification products
after each cycle, and (iii) Calculating the ratio between the
amount of DNA from the first gene locus and the amount of DNA from
the second gene locus. The new method is useful for a variety of
applications, especially for detection of chromosomal abnormalities
in fetal cells.
Inventors: |
Costa, Jean-Marc; (Paris,
FR) |
Correspondence
Address: |
ROCHE MOLECULAR SYSTEMS INC
PATENT LAW DEPARTMENT
1145 ATLANTIC AVENUE
ALAMEDA
CA
94501
|
Family ID: |
8176348 |
Appl. No.: |
10/470986 |
Filed: |
January 23, 2004 |
PCT Filed: |
January 29, 2002 |
PCT NO: |
PCT/EP02/00879 |
Current U.S.
Class: |
435/6.11 ;
435/6.17; 435/91.2 |
Current CPC
Class: |
C12Q 1/6879 20130101;
C12Q 1/686 20130101; C12Q 1/6883 20130101; C12Q 2545/113 20130101;
C12Q 2545/107 20130101; C12Q 2545/114 20130101; C12Q 1/686
20130101; C12Q 1/6881 20130101; C12Q 2600/156 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 31, 2001 |
EP |
01102106.0 |
Claims
We claim:
1. Method for genotype determination at a specific gene locus of an
individual or a fetus comprising a) Amplifying a first sequence of
said gene locus and a second sequence of a second reference gene
locus from DNA originating from a sample containing biological
material of said individual or fetus b) Quantifying the original
amount of DNA of said first gene locus and said second gene locus
present in the sample c) Calculating the ratio between the amount
of DNA from the first gene locus and the amount of DNA from the
second gene locus originally present in the sample prior to the
amplification reaction
2. Method for genotype determination at a specific gene locus of an
individual or a fetus comprising a) Amplifying a first sequence of
said gene locus and a second sequence of a second reference gene
locus from DNA originating from a sample containing biological
material of said individual or fetus b) Monitoring both
amplifications in real time and determining the amount of
amplification products after each cycle c) Calculating the ratio
between the amount of DNA from the first gene locus and the amount
of DNA from the second gene locus
3. Method for genotype determination at a specific gene locus of a
fetus comprising a) Isolating fetal cells, preferably by
Immuno-enrichment b) Amplifying a first sequence of said gene locus
and a second sequence of a second reference gene locus from DNA
originating from cells isolated in step a) c) Monitoring both
amplifications in real time and determining the amount of
amplification products after each cycle d) Calculating the ratio
between the amount of DNA from the first gene locus and the amount
of DNA from the second gene locus originally present in the sample
prior to the amplification reaction method
4. Method according to claim 1-3, wherein said first and said
second sequence are amplified and detected in one tube
5. Method according to claim 1-4, wherein the amplification
products are detected using fluorescent signals
6. Method according to claim 5, wherein the amplification probes
are detected with FRET/Hybridization probes
7. Method according to claims 1-6, wherein either said specific
gene locus or said reference gene locus is MBP or SOD.
8. Use of a method according to claims 1-7, wherein either said
specific gene locus or said reference gene locus is the RhD
gene.
9. Use of a method according to claims 1-7 for detecting
chromosomal abnormalities
10. Use according to claim 9 for determination of trisomy 21,
trisomy 18, or trisomy 13
11. Use according to claim 9 for determination of microdeletional
syndromes
12. Use according to claim 9 for determination of a female carrier
status for a Chromosome X-linked genetic disorder
13. Use according to claim 9, wherein said genetic disorder is
Heamophilia or Myopathy
14. Use of a method according to claims 1-7, for sex determination
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to the field of genotype
determination. More specifically, the new invention relates to the
field of determination of genotypes using nucleic acid
amplification technologies like the Polymerase Chain Reaction
(PCR).
DESCRIPTION OF RELATED ART
[0002] Determination of a certain genotype of an individual
sometimes may be a an essential diagnostic tool in order to decide
on the medical treatment of a patient, e.g. with regard for his
susceptibility for a certain therapeutic drug.
[0003] Especially for prenatal diagnostics, genotype determination
is performed frequently. For example, in case pregnant women are
homozygously negative with respect to the RhD blood group system
antigen, it is important to determine the fetal RhD genotype, since
RhD-heterozygous babies from RhD-homozygously negative mothers
could suffer from allo-immunization reactions and hemolytic anemia,
in case no prophylactic treatment is taking place (Whittle, Arch
Dis Child 1992 January;67(1 Spec No):65-8). Another important field
is the test for trisomy 21 resulting in Down syndrome, trisomy 18
resulting in Edwards syndrome and trisomy 13 resulting in Patau
syndrome, since there exists an increased risk in case the pregnant
women are older than 35 years.
[0004] In order to perform genotyping on fetal DNA, fetal cells are
usually obtained by amniocentesis and subjected to further
investigation. Alternatively, few fetal cells may be collected from
the mother's blood stream, which, however, usually need to be
enriched by immunological or physical methods prior to subsequent
genotyping experiments (Pertl et al, Semin Perinatol 1999
October;23(5):393-402). Traces of free floating fetal DNA has also
been observed in the blood stream of the mother (Lo et al, Lancet.
1997 Aug. 16;350(9076):485-7). However, due to the presence of
maternal DNA background, a genotype analysis regarding the fetal
DNA is only possible in very specific cases like RhD in the
background of a RhD recessive mother (Lo et al., New England J. of
Medicine 339, p. 1734-1738, 1998), but not for other cases like
e.g. Trisomy 13, 18, or 21.
[0005] In the past, genotype determination usually has been
performed by means of conventional microscopy, which requires a
labour extensive and sophisticated chromosomal spreading.
Eventually, these methods have been combined with in situ
hybridization protocols like the FISH technique (Philip et al,
Prenat Diagn 1994 Dec;14(13):1203-15). Alternatively, DNA Southern
Blot based molecular methods may now be applied. These include, for
example, conventional Restriction Fragment Length Polymorphism
analysis. However, due to the limited amount of available sample
material, these methods are not applicable for prenatal
diagnostics.
[0006] Among the number of different analytical methods that detect
and quantify nucleic acid sequences, Polymerase Chain Reaction
(PCR) has become the most powerful and wide-spread technology, the
principles of which are disclosed in U.S. Pat. No. 4,683,195 and
U.S. Pat. No. 4,683,102 (Mullis et al.). However, a typical PCR
reaction by itself only yields qualitative data, since, after a
phase of exponential or progressive amplification, the amount of
amplified nucleic acid reaches a plateau, such that the amount of
generated reaction product is not proportional to the initial
concentration of the template DNA.
[0007] As a consequence, many different PCR based protocols have
been developed in order to obtain reliable and reproducible
quantitative data. Generally, three different basic principles can
be discriminated:
[0008] i) Relative Quantification of a target nucleic acid compared
to a standard within the sample (e.g. expression of a housekeeping
gene in case of RT-PCR)
[0009] ii) Quantification with competitive PCR using an internal
standard
[0010] iii) Quantification of target DNA using an external
standard
[0011] A major improvement in the generation of quantitative data
derives from the possibility of measuring the kinetics of a PCR
reaction by On-Line detection. This has become possible recently by
means of detecting the amplicon through fluorescence monitoring.
Examples of such techniques are disclosed in detail in WO 97/46707,
WO 97/46712 and WO 97/46714 (Wittwer et al.), the disclosures of
which are hereby incorporated by reference.
[0012] All these methods can easily be designed as assays with a
dynamic range of less than about 2 orders of magnitude, i.e.
wherein the concentrations of the target nucleic acids in two
different samples to be determined do not differ more than a
hundred fold. However, regarding the lower discrimination limit,
prior to the new invention no reliably protocols have been
published, which disclose the discrimination of concentration
differences between two target concentrations that only differ from
each other by not more than 50%.
[0013] Several detection formats based on target nucleic acid
dependent fluorescent signaling have been disclosed, which enable
continuous monitoring of the generation of amplification products
(reviewed in Wittwer et al., Biotechniques, Vol. 22, No, 1,
130-138, 1997). These detection formats include but are not limited
to:
[0014] 1. Use Of Fluorescent Double-Stranded DNA Recognizing
Compounds
[0015] Since the amount of double stranded amplification product
usually exceeds the amount of nucleic acid originally present in
the sample to be analyzed, double-stranded DNA specific dyes may be
used, which upon excitation with an appropriate wavelength show
enhanced fluorescence only if they are bound to double-stranded
DNA. Preferably, only those dyes may be used which like SYBR Green
I (Molecular Probes), for example, do not affect the efficiency of
the PCR reaction.
[0016] 2. Increased Fluorescence Resonance Energy Transfer Upon
Hybridization
[0017] For this detection format, two oligonucleotide hybridization
probes each labeled with a fluorescent moiety are used which are
capable of hybridizing to adjacent but non overlapping regions of
one strand of the amplification product. Preferably, one
oligonucleotide is labeled at the 5' end and the second
oligonucleotide is labeled at the 3' end. When hybridized to the
target DNA, the two fluorescent labels are brought into close
contact, such that fluorescence resonance energy transfer between
the two fluorescent moieties can take place. As a consequence, the
hybridization can be monitored through excitation of the donor
moiety and subsequent measurement of fluorescence emission of the
second acceptor moiety.
[0018] In a similar embodiment, only one fluorescently labeled
probe is used, which together with one appropriately labeled primer
may also serve as a specific FRET pair (Bernard et al., Analytical
Biochemistry 235, p. 101-107 (1998)).
[0019] 3. Taq Man Principle
[0020] In order to detect the amplification product, a
single-stranded hybridization probe is used, which is labeled with
a fluorescent entity, the fluorescence emission of which is
quenched by a second label on the same probe which may act as a
quenching compound. During the annealing step of the PCR reaction,
the probe hybridizes to its target sequence, and, subsequently,
during the extension of the primer, the DNA polymerase having a
5'-3'-exonuclease activity digests the hybridization probe into
smaller pieces, such that the fluorescent entity is separated from
the quencher compound. After appropriate excitation, fluorescence
emission can be monitored as an indicator of accumulating
amplification product.
[0021] 4. Molecular Beacons
[0022] Similar to the Taq Man Probes, a molecular beacon
oligonucleotide is labeled with a fluorescent compound and a
quencher compound, which due to the secondary structure of the
molecule are in dose vicinity to each other. Upon binding to the
target DNA, the intramolecular hydrogen bonding is broken, and the
fluorescent compound located at one end of the probe is separated
from the quencher compound, which is located at the opposite end of
the probe (Lizardi et al., US. Pat. No. 5,118,801).
[0023] All the prior art methods discussed above, which may be used
for the assessment of an allelic status, however, show major
disadvantages:
[0024] Determination of an allelic status using microscopic
techniques requires time consuming chromosomal spreading and
staining procedures
[0025] Southern Blot based hybridization methods like Restriction
Fragment Length Polymorphism require a large amount of starting
material, which especially in prenatal diagnosis is not
obtainable.
[0026] Conventional qualitative prior art Nucleic Acid
amplification methods like PCR are useful in order to detect the
presence or absence of a specific allele, however, these methods
are not suited in order to discriminate between a homo-or
heterozygous status or detect the existence of low copy allelic
amplification.
[0027] Conventional quantitative prior art Nucleic Acid
amplification methods require an extensive calibration and can not
easily discriminate between one, two or three copies of a target
nucleic acid originally present in the sample.
SUMMARY OF THE INVENTION
[0028] Therefore, there exists a need in the art for a fast and
easy method which allows for a rapid determination of an allelic
status of a specific gene locus or even a complete chromosome
starting from a limited amount of sample material.
[0029] Thus, the new invention is directed to a method for genotype
determination at a specific gene locus of an individual or a fetus
comprising
[0030] a) Amplifying a first sequence of said gene locus and a
second sequence of a second reference gene locus from DNA
originating from a sample containing biological material of said
individual or fetus
[0031] b) Quantifying the original amount of DNA of said first gene
locus and said second gene locus present in the sample
[0032] c) Calculating the ratio between the amount of DNA from the
first gene locus and the amount of DNA from the second gene locus
originally present in the sample prior to the amplification
reaction
[0033] Thus, the ratio is indicative for the genotype of the sample
to be analyzed. More specifically, the invention is directed to a
method for genotype determination at specific gene locus of an
individual or a fetus comprising
[0034] a) Amplifying a first sequence of said gene locus and a
second sequence of a second reference gene locus from DNA
originating from a sample containing biological material of said
individual or fetus
[0035] b) Monitoring both amplifications in real time and
determining the amount of amplification products after each
cycle
[0036] c) Calculating the ratio between the amount of DNA from the
first gene locus and the amount of DNA from the second gene
locus
[0037] In a major aspect of the invention, a sample of fetal cells
is used, which has been obtained either from or amniocentesis or
from maternal blood. In the latter case, it is preferred if the
fetal cells have been isolated by immuno-enrichment or physical
enrichment.
[0038] In the context of the new method, it has been proven to be
advantageous, if the first and the second sequence are amplified
and detected in one tube.
[0039] Detection of the amplification products is preferably
obtained by means of detecting fluorescent signals and most
preferably by means of using a couple of FRET/Hybridization
probes.
[0040] One specific embodiment of the invention is directed to the
methods disclosed above, wherein the MBP gene locus, the SOD gene
locus or the RhD gene locus are amplified and analyzed.
[0041] It is within the scope of the present invention to use the
claimed method in order to detect chromosomal abnormalities. Among
these abnormalities, chromosomal aberrations like trisomy 21,
trisomy 18, trisomy 13 or microdeletional syndromes may be detected
according to the invention.
[0042] The new invention may also be used to determine a female
carrier status for a Chromosome X-linked genetic disorder like for
example, Haemophilia or Myopathy. Moreover, the new method may also
be used for sex determination, if DNA from fetal cells is
analyzed.
DETAILED DESCRIPTION OF THE INVENTION
[0043] In a first and predominant aspect, the present invention
provides a general method for genotype determination at a specific
gene locus of an individual or a fetus comprising
[0044] a) Amplifying a first sequence of said gene locus and a
second sequence of a second reference gene locus from DNA
originating from a sample containing biological material of said
individual or fetus
[0045] b) Quantifying the original amount of DNA of said first gene
locus and said second gene locus present in the sample
[0046] c) Calculating the ratio between the amount of DNA from the
first gene locus and the amount of DNA from the second gene locus
originally present in the sample prior to the amplification
reaction
[0047] Amplification of said first and second sequence is usually
performed by means of PCR. Quantification of the original amount of
DNA can be achieved by any method known in the art such as
application of an external standard or application of an internal
standard for competitive PCR. In the latter case, the internal
standard is usually amplified with the same primers like the target
nucleic acid itself. Preferably, the sequence of the internal
competitor is similar to the sequence of the target nucleic acid,
such that both are amplified with about the same efficiency. It is
also within the scope of the invention, in case the amount of
target DNA is not determined in an absolute value rather than as a
relative value as compared to the external standard or the
competitor DNA without knowing the actual concentration of the
target- or competitor DNA itself.
[0048] In the context of this invention, genotype determination is
understood as determination of the gene dosis of a specific allele
present in the genome to be analyzed. According to the invention,
concentrations of amplification products originating from 2
different gene loci are always compared with each other. As will be
shown in the examples, the new method is able to identify
differences between one and two copy numbers of an allele (for
example in case of of RhD and Factor VIII), or even between two or
three copies of an allele (for example in case of trisomy
detection). In the latter case, the new invention provides a method
in order to discriminate differences in gene dosage of as few as
50%.
[0049] The term "individual" in the context of this invention not
only comprises an individual human being, but also an individual
animal or plant specimen. Furthermore, individual may also mean a
particular strain of a microorganism, originating from one single
clone.
[0050] The present invention is especially directed to a method for
genotype determination at a specific gene locus of an individual or
a fetus comprising
[0051] a) Amplifying a first sequence of said gene locus and a
second sequence of a second reference gene locus from DNA
originating from a sample containing biological material of said
individual or fetus
[0052] b) Monitoring both amplifications in real time and
determining the amount of amplification products after each
cycle
[0053] c) Calculating the ratio between the amount of DNA from the
first gene locus and the amount of DNA from the second gene
locus
[0054] In this preferred embodiment, the assay is performed in a
homogeneous detection format in Real Time. That means, a suitable
hybridization probe is already present during the amplification
reaction. The hybridization probe preferably carries a fluorescent
label which is detectable after appropriate excitation. Due to the
possibility of kinetic measurements during the amplification
itself, monitoring the reaction in Real Time strongly facilitates
the quantification of the target DNAs. Again all methods and
instruments known in the art for Real Time PCR quantification may
be used.
[0055] In this context, however, it is important to know, that the
signals obtained by Real Time PCR do not exclusively reflect the
amount of target DNAs present in the sample, since the intensity of
the signals is also dependent on the sensitivity of the detection
system applied. The sensitivity itself is influenced by several
parameters like e.g. melting point of the specific hybridization
probe or fluorescent quantum yield. As a consequence, for example,
homozygous existence of a first allele compared to heterozygous
existence of a second reference allele in this type measurements
does not exactly result in a ratio of 2:1. Nevertheless, the method
according to the new invention allows for discrimination between a
homozygous and a heterozygous state of the same allele (see
examples below).
[0056] A specific aspect of the invention is directed to a method
for genotype determination at a specific gene locus of a fetus
comprising
[0057] a) Isolating fetal cells preferably by Immuno-enrichment or
physical enrichment
[0058] b) Amplifying a first sequence of said gene locus and a
second sequence of a second reference gene locus from DNA
originating from cells isolated in step a))
[0059] c) Monitoring both amplifications in real time and
determining the amount of amplification products after each
cycle
[0060] d) Calculating the ratio between the amount of DNA from the
first gene locus and the amount of DNA from the second gene locus
originally present in the sample prior to the amplification
reaction method
[0061] In case a prenatal analysis is performed, it is advantageous
either to use cells obtained by amniocentesis or, alternatively to
enrich fetal cells obtained from maternal blood by immunological
methods, preferably with an antibody that binds to a surface
antigen of a fetal cell, which is not expressed on the surface of
cells found in the maternal blood vessel system (Wang et al,
Cytometry 2000 Mar. 1;39(3):224-30). In a still further alternative
approach, fetal cells may be enriched by a Ficoll gradient
centrifugation according to the teaching of (Samura et al, Prenat
Diagn 2000 April;20(4):281-6)
[0062] One possibility for Immuno-enrichment is the use of a laser
based cell sorting system. In this case, the cell surface is
contacted with a fluorescently labeled antibody. The cell/antibody
complex is then subjected to a cell sorter, which is able to
separate labeled from unlabeled cells (Sekizawa et al, Fetal Diagn
Ther 1999 July-August;14(4):229-33). Alternatively, the cells may
be isolated through affinity binding to an antibody which has
previously been immobilized onto a solid support.
[0063] A person skilled in the art will recognize that different
detection formats may be applied in order to perform the claimed
genotype determination according to the invention. Most commonly in
Real Time PCR, the amplification products are detected using
fluorescent signals. This is possible by detecting the
amplification products with a ds DNA binding fluorescent Dye such
as Ethidium Bromide, SybrGreen or SybrGold (Molecular Probes).
[0064] Alternatively, fluorescently labeled hybridization probes
may be used. Independent from the detection format or fluorescent
label, Hybridization probes are always polynucleotides having
sequences which are completely identical with or exactly
complementary to the sequence of the target nucleic acid. Yet, it
is also within the scope of the invention, if the probes contain
one or several mismatches, as long as they are capable of
hybridizing to the analyte under appropriate hybridization
conditions. In any case, it has been proven to be particular
advantageous, if the sequence identity or complementarity is 100%
over a range of at least 10 contiguous residues. It has also been
proven to be advantageous, if the length of the probe does not
exceed 100 nucleotides, preferably not more than 40 nucleotides.
However, hybridization probes may have 5' or 3' overhangs which do
not hybridize to the target nucleic acid.
[0065] The term "Polynucleotide" in this context summarizes not
only (Desoxy)-Oligo-Ribonucleotides, but also all DNA-or
RNA-derivatives known in the art like e.g. Methyl-Phosphonates,
Phosphothioates, 2'-O-Alkyl-derivatives as well as Peptide Nucleic
Acids, and analoga comprising modified bases like
7-Deaza-Purines.
[0066] Hybridization probes such as TaqMan or Molecular beacons may
be used. Most preferred are FRET/Hybridization probes, i.e. a pair
of adjacently hybridizing probes, wherein upon hybridization the
two fluorescent moieties are brought into close vicinity such that
Fluorescent Resonance Energy Transfer can take place. The term,
FRET Hybridization probes" therefore is defined as a pair of
hybridization probes, each probe carrying a fluorescent compound,
which together may act as a FRET pair thus enabling the detection
of a nucleic acid, when both probes are hybridized adjacently to a
target molecule.
[0067] According to the invention, it is preferred, if the first
and the second target sequence are amplified and detected in one
tube, since quantification errors due to variable amounts of
starting materials can be excluded. This is possible in a multiplex
approach, wherein differentially labeled hybridization probes for
each sequence are used for detection of the respective
amplification products.
[0068] Such assays may be performed on a Light Cycler instrument
(Roche Molecular Biochemicals) using a first pair of FRET
Hybridization probes labeled with Fluorescein at the 3' end of the
first oligonucleotide and with LC-Red-640 (Roche Molecular
Biochemicals) at the 5' end of the second oligonucleotide and a
second pair of FRET Hybridization probes labeled with Fluorescein
at the 3' end of the first oligonucleotide and with LC-Red-705
(Roche Molecular Biochemicals) at the 5' end of the second
oligonucleotide.
[0069] Principally, any kind of quantification method can be
applied, however, it has been proven to be advantageous, if methods
using an external standard are applied. The external standard
itself may either be a plasmid or a linearized template with the
target sequences to be amplified or, alternatively, genomic DNA
wherein the phenotypes of the gene loci to become investigated.
[0070] In case of quantification of a nucleic acid using external
standards, a calibration curve has to be generated. For this
calibration curve, known amounts of the target nucleic acid are
amplified and the intensity of fluorescent signal is determined as
a function of cycle number. After smoothening of the kinetics by a
mathematical fit, the first or second maximum of the derivative are
calculated. This enables a correlation between the original target
concentration and the fractional cycle number of a determined
maximum. Subsequently, determination of unknown analyte
concentrations may be performed.
[0071] In order to eliminate quantification errors originating from
different detection sensitivities, it has been proven to be
particular advantageous, if the same batch of hybridization
probe(s) is used for the sample to be analyzed and for the
calibration samples.
[0072] The claimed method can be used for analysis of multiple
different gene loci as well as for the analysis of chromosomal
abnormalities like whole chromosome aberrations, e.g the detection
of trisomy disorders like trisomy 13, 18, or 21. The list of
applications, however, is not restricted to the examples given
below.
[0073] The reference gene always can be chosen almost arbitrarily,
as long as it is possible to establish a quantitative and
reproducible amplification reaction with respect to this target.
Notwithstanding the foregoing, the reference allele should of
course be reasonable stable and invariant within a given
population. For analysis of whole chromosome aberrations, it is
clear that the reference gene needs to be located on a chromosome
different from the one with the allele to be investigated.
[0074] The genes encoding Superoxide Dismutase (SOD) located on
Chromosome 21 and Myelin Basic protein (MBP) located on Chromosome
18 have been proven to be particular advantageous not only as
reference genes but also as indicator alleles for detection of
chromosomal abnormalities for trisomy 18 or trisomy 21. Even more
preferred embodiments comprise the usage of one or more primers
according to Seq. Id. No: 1-4, which may be detected by
FRET-Hybridization probes according to Seq. Id. No. 5-6 for SOD and
7-8, respectively for MBP.
[0075] In another aspect, the invention is directed to the
determination of the allelic status of RhD. This is preferably done
on parental DNA, which is extremely important in case the mother is
RhD negative. However, the assay can also be performed on fetal
DNA. Appropriate primers are Oligonucleotides according to Seq. Id.
No. 9-10. FRET-Hybridization probes according to Seq. Id. No. 11-12
may be used. SOD, MBP or any other target may be chosen as a
reference gene.
[0076] Another aspect of the invention is directed to the
determination of a female carrier status of a Chromosome X-linked
genetic disorder like Heamophilia (Factor VIII) or Myopathy. For
Heamophila, appropriate primers are Oligonucleotides according to
Seq. Id. No. 13-14. FRET-Hybridization probes according to Seq. Id.
No. 15-16 may be used. Again, SOD or MBP preferably may serve as
reference genes. Yet another aspect of the invention is directed to
sex determination based on analysis of fetal DNA using the X-linked
SRY gene and a second autosomal gene as reference alleles.
[0077] The following examples, references and sequence listing are
provided to aid the understanding of the present invention, the
true scope of which is set forth in the appended claims. It is
understood that modifications can be made in the procedures set
forth without departing from the spirit of the invention.
EXAMPLES
[0078] Harvest and Culturing of Amniotic Fetal Cells
[0079] Amniotic fetal cells are harvested from amniotic fluid by
amniocentesis performed as soon as 14 weeks of pregnancy. The cells
are obtained from 10 ml of AF after centrifugation (5 min. at 1000
rpm/min) and are processed in the following manner;
[0080] cells are resuspended in 5 ml of supernatant 1 ml of this
suspension is added to in a cell culture medium (ie HAM F10) and
then incubated at 37.degree. C. with 5% CO2 in a incubator.
Example 1
DNA Sample Preparation from Fetal Cells
[0081] For preparation of fetal genomic DNA from cultured cells,
the HighPure PCR template preparation kit was used (Roche Molecular
Biochemicals, Cat. No. 1796 828) which is based on cell lysis by
means of adding Guanidinium-HCL.
Example 2
Real Time PCR Amplification
[0082] LightCycler PCRs were set up in a final volume of 20 .mu.l
with the FastStart DNA Master Hybridization Probes Kit (Roche
Molecular Biochemicals), each primer at a concentration of 0,5
.mu.M, each probe at a concentration of 0,25 .mu.M and 5 .mu.l of
extracted DNA sample corresponding to about 10.sup.+3 cells, which
had been isolated according to example 1. A hot-start procedure was
systematically applied. Carryover contamination was prevented using
heat-labile Uracil-DNA-Glycosylase (UNG, Roche Molecular
Biochemicals). The reaction mix is listed in table 1 below:
1TABLE 1 Volume [.mu.l] [Final] LC Fast-DNA Master Hybridization
Probes 2 1x (including FastStartTaq-Polymerase) MgCl.sub.2 (25 mM)
3.2 5 mM Primers (20 .mu.M each) Target Gene 0.5 0.5 .mu.M
Hybridization probes LCRed 640 (20 .mu.M 0.25 0.25 .mu.M each)
Primers (20 .mu.M each) Reference Gene 0.5 0.5 .mu.M Hybridization
probes LCRed705 (20 .mu.M 0.25 0.25 .mu.M each) Uracil-DNA
glycosylase (1 U/.mu.l) 0.25 0.25 U Standard DNA in appropriate
dilution 5 H.sub.20 (PCR grade) 8.05 Total volume 20
[0083] For unknown samples, replace the standard DNA with sample
DNA extract.
[0084] The reaction mixture was initially incubated for 1 min at
room temperature to allow UNG to act. This incubation was followed
by a 8-min step at 95.degree. C. to denature the DNA, to inactivate
UNG, and to activate Taq DNA polymerase. Amplification was
performed in a LightCycler (Roche Molecular Biochemicals) according
to the following temperature cycling protocol:
[0085] Denaturation: 95.degree. C. for 10 s (ramp rate 20.degree.
C./s)
[0086] Annealing: 60.degree. C. for 10 s (ramp rate 20.degree.
C./s)
[0087] Extension: 72.degree. C. for 15 s (ramp rate 2.degree.
C./s)
[0088] During thermocycling, a single fluorescence reading for each
sample was taken at the annealing step. Quantitative results were
expressed by determination of the crossing point Cp which marked
the cycle when the measured fluorescent signal exceeded a certain
threshold of signal intensity. Subsequently, the Cp's were plotted
against an external standard calibration curve of known
concentrations of the target nucleic acid, which had been amplified
and measured previously. Genomic DNA with known phenotype always
served as an external standard.
Example 3
Detection of Trisomy 21 and 18
[0089] Using the SOD gene as a marker for Chromosome 21 and the MBP
gene as a marker for Chromosome 18, an analysis for trisomy 18 and
21 was performed such that trisomy 21 was expected to result in an
increased gene dosage of SOD and trisomy 18 was expected to result
in an increased dosage of MBP.
[0090] Sample preparation was performed according to example 2.
Real time PCR was carried out according to example 2.
[0091] For amplification and detection of SOD, primers according to
Seq. Id. No: 1 and 2 and FRET-Hybridization probes according to
Seq. Id. No. 5 (labeled with Fluorescein at its 3' end) and Seq.
Id. No: 6 (labeled with LC-Red-640 at its 5' end) were used. For
amplification and detection of MBP, primers according to Seq. Id.
No: 3 and 4 and FRET-Hybridization probes according to Seq. Id. No.
7 (labeled with Fluorescein at its 3' end) and Seq. Id. No: 8
(labeled with LC-Red-705 at its 5' end) were used. Amplification of
both targets was carried out in a single tube, however, each sample
was amplified and measured at least in duplicate. All results with
respect to the determined Genotype were confirmed by conventional
Caryotype analysis using conventional techniques of chromosomal
spreading and light microscopy. The obtained data from 2
independent experiments resulting in different detection
sensitivities are listed in Table 2.
2TABLE 2 SOD MBP Ratio Sample No. ng/.mu.l ng/.mu.l SOD:MBP
Caryotype 1 204 97 2.11 46 + Chr 21 2 1235 862 1.43 46 + Chr 21 3
726 985 0.74 46 4 21 64 0.32 46 + Chr 18 5 1177 853 1.38 46 + Chr
21 6 1412 1767 0.80 46 7 10.67 7.27 1.47 46 + Chr 21 8 1.95 2.60
0.75 46 + Chr 18 9 3.45 3.95 0.87 46 10 15.42 11.79 1.31 46 + Chr
21 11 1.39 1.28 1.09 46 12 9.45 8.45 1.21 46 + Chr 21 13 3.46 3.80
0.91 46
[0092] The data of this table 2 are from 2 independent experiments
with different batches of hybridization probes; samples 1-6 were
assayed in the first experiment, and samples 7-13 were assayed in
the second experiment. As shown, a normal genotype resulted in a
SOD/MBP ratio between 0, 74 and 0,80 in the first experiment and a
ratio between 0,87 and 1,09 in the second experiment. A trisomy 21
genotype resulted in a SOD/MBP ratio of at least 1.21 for both
experiments. A trisomy 18 genotype resulted in a SOD/MBP ratio of
about 0.32 in the first experiment and a ratio of 0.75 in the
second experiment. These data clearly demonstrate that the
disclosed method is applicable for diagnosis of trisomies, as soon
as enough material from the individual to be analyzed is available
in order to perform a typical Real Time PCR experiment, provided,
however, that intra assay variances are excluded.
Example 4
Determination of RhD Genotype
[0093] DNA from 101 individuals was basically analyzed as disclosed
in example 3. For amplification and detection of RhD, primers
according to Seq. Id. No: 9 and 10 and FRET-Hybridization probes
according to Seq. Id. No. 11 (labeled with Fluorescein at its 3'
end) and Seq. Id. No: 12 (labeled with LC-Red-640 at its 5' end)
were used. MBP was detected as a reference gene in the same samples
using primers and FRET-Hybridization probes according to example
3.
[0094] For this approach, the RhD/MBP ratio was expected to be
increased in case of a homozygous DD genotype as compared to a
heterozygous Dd genotype, whereas in case of a homozygous dd
genotpype resulting from a complete deletion of the RhD gene,
amplification of RhD does not occur. All results with respect to
the determined Genotype/Phenotype were confirmed by a conventional
RhD agglutination assay (Diamed). The obtained data are listed in
table 3:
3TABLE 3 Ratio Phenotype in Sample No. RhD ng/.mu.l MBP ng/.mu.l
MBP/RhD agglutination assay 1 4.1 4.7 1.15 DD 2 18.5 21.3 1.15 DD 3
17.3 20.1 1.16 DD 4 8.9 10.4 1.17 DD 5 21.5 25.5 1.19 DD 6 24.2
28.9 1.19 DD 7 12.3 14.7 1.20 DD 8 17.1 20.7 1.21 DD 9 47.8 58.1
1.22 DD 10 6 7.3 1.22 DD 11 31.2 38.0 1.22 DD 12 22 27 1.23 DD 13
23.7 29.1 1.23 DD 14 21 25.8 1.23 DD 15 22.6 27.8 1.23 DD 16 15
18.5 1.23 DD 17 22.5 27.8 1.24 DD 18 36.5 45.2 1.24 DD 19 17.6 22
1.25 DD 20 30.2 37.9 1.25 DD 21 15.9 20 1.26 DD 22 22.4 28.3 1.26
DD 23 38.6 49 1.27 DD 24 18.1 23 1.27 DD 25 22.1 28.1 1.27 DD 26
20.6 26.2 1.27 DD 27 45.7 58.2 1.27 DD 28 45.1 57.5 1.27 DD 29 30.1
38.4 1.28 DD 30 12.3 15.7 1.28 DD 31 13.2 16.9 1.28 DD 32 23.9 30.6
1.28 DD 33 10.4 13.4 1.29 DD 34 20.6 26.8 1.30 DD 35 28.5 37.7 1.32
DD 36 20.1 26.6 1.32 DD 37 8.9 11.8 1.33 DD 38 29.2 39 1.34 DD 39
14.7 19.7 1.34 DD 40 18.3 24.8 1.36 DD 41 14.2 19.4 1.37 DD 42 22.6
31.5 1.39 DD 43 12.1 17 1.40 DD 44 14 19.9 1.42 DD 45 18.9 27 1.43
DD 46 14.6 22.2 1.52 DD 47 53 80.6 1.52 DD 48 11.4 25.2 2.21 Dd 49
8.5 20 2.35 Dd 50 16.8 39.6 2.36 Dd 51 11.2 26.5 2.37 Dd 52 9.7
23.1 2.38 Dd 53 5.9 14.1 2.39 Dd 54 15.8 37.9 2.40 Dd 55 17.3 41.5
2.40 Dd 56 3.5 8.4 2.40 Dd 57 16.9 40.6 2.40 Dd 58 10.2 24.4 2.40
Dd 59 7.5 18.1 2.41 Dd 60 2.9 7 2.41 Dd 61 11.3 27.4 2.42 Dd 62
10.8 26.2 2.43 Dd 63 12.6 32.1 2.55 Dd 64 10.5 26.8 2.55 Dd 65 17.6
45 2.56 Dd 66 11 28.2 2.56 Dd 67 32.7 84.1 2.57 Dd 68 9.4 24.2 2.57
Dd 69 15.3 39.5 2.58 Dd 70 8.4 21.7 2.58 Dd 71 13.6 35.2 2.59 Dd 72
6.1 15.8 2.59 Dd 73 8.5 22.1 2.60 Dd 74 12.7 33.1 2.61 Dd 75 22.1
57.6 2.61 Dd 76 7.3 19.1 2.62 Dd 77 7.2 18.9 2.63 Dd 78 3.5 9.2
2.63 Dd 79 12.3 32.5 2.64 Dd 80 20.0 54.0 2.70 Dd 81 16.8 45.4 2.70
Dd 82 24.5 67.1 2.74 Dd 83 12.3 33.7 2.74 Dd 84 5.1 14 2.75 Dd 85
24.7 67.9 2.75 Dd 86 11.2 30.9 2.76 Dd 87 6.4 17.8 2.78 Dd 88 6.8
19.3 2.84 Dd 89 26.2 74.4 2.84 Dd 90 6.5 18.6 2.86 Dd 91 10.6 31.1
2.93 Dd 92 NEG ok Dd 93 NEG OK Dd 94 NEG ok Dd 95 NEG ok Dd 96 NEG
Ok Dd 97 NEG ok Dd 98 NEG ok Dd 99 NEG ok Dd 100 NEG Ok Dd 101 NEG
Ok Dd
[0095] As shown in Table 3, a DD genotype resulted in a MBP/RhD
ratio of about 2,57 with a standard deviation of +/-0,17. A Dd
genotype resulted in a MBP/RhD ratio of about 1,28 with a standard
deviation of about +/-0,09, whereas amplification could not be
observered in case of the dd genotype. These data clearly show that
using this method, the RhD genotype can be determined reliably.
Example 5
Determination of Haemophilia Carrier Status
[0096] DNAs from 31 individuals were analyzed basically according
to example 3. Due to occuring Haemophilia in male member of their
family (No. 26), 3 females (No. 29,30, and 31) were suspected to
have a heterozygous Factor VIII molecular defect resulting in a
Haemophilia carrier status.
[0097] Analysis was principally performed as disclosed in example
4. For amplification and detection of Factor VIII, exon 1, primers
according to Seq. Id. No: 13 and 14 and FRET-Hybridization probes
according to Seq. Id. No. 15 (labeled with Fluorescein at its 3'
end) and Seq. Id. No: 16 (labeled with LC-Red-705 at its 5' end)
were used. SOD was detected as a reference gene in the same samples
using primers and FRET-Hybridization probes according to example
3.
[0098] For this approach, the FactorVIII/SOD ratio in female
carriers was expected to be different to the ratio observed in wild
type female individuals but similar to male individuals, since both
male individuals and female carriers carry one intact copy of the
Factor VIII gene. The obtained data are listed in table 4. The
table shows the values of two independendent experiments using
different batches of hybridizazion probes. The different
experiments are indicated by sample numbers 1-25 and 26-31.
4TABLE 4 Sample Factor VIII SOD Ratio SEX No. Ng/.mu.l ng/.mu.l
SOD:Factor VIII Carrier (c) 1 24.8 24.7 1.00 XX 2 27.7 28.1 1.01 XX
3 17.7 18.7 1.06 XX 4 13.7 26.4 1.93 XY 5 65.8 74.3 1.13 XX 6 8.6
18.0 2.09 XY 7 39.1 40.5 1.04 XX 8 27.1 28.5 1.05 XX 9 38.2 41.2
1.08 XX 10 14.3 28.9 2.02 XY 11 23.9 22.7 0.95 XX 12 7.9 15.6 1.97
XY 13 72.3 76.5 1.06 XX 14 46.1 48.9 1.06 XX 15 23.9 48.3 2.02 XY
16 22.1 43.9 1.99 XY 17 34.8 39.1 1.12 XX 18 11.7 22.2 1.90 XY 19
19.2 38.3 1.99 XY 20 37.4 38.5 1.03 XX 21 47.2 43.4 0.92 XX 22 11
20.8 1.89 XY 23 13.5 26.4 1.96 XY 24 37.2 40.2 1.08 XX 25 30.2 30.9
1.02 XX 26 Neg/Del 15.9 #VALUE! XY, Haemophillic 27 6.9 15.5 2.25
XX 28 21 52.3 2.49 XX 29 19.4 15.5 0.80 XX, Carrier 30 18.8 16.2
0.86 XX, Carrier 31 14.2 11.5 0.81 XX, carrier
[0099] As it can be deduced from the table, in each of the two
independent experiments, two clear non overlapping populations can
be identified: a) values found for female individuals definitely
carrying two alleles of Factor VIII, and b) values for male
individuals carrying one allele. The ratios obtained for the two
female suspected to be haemophilia carrriers were indeed similar to
the values obtained for male individuals suggesting that they are
Haemophilia carriers.
LIST OF REFERENCES
[0100] Bernard, et al., Analytical Biochemistry 235, p. 101-107
(1998)
[0101] Lizardi et al., U.S. Pat. No. 5,118,801
[0102] Lo et al, Lancet. 1997 Aug. 16;350(9076):485-7
[0103] Lo et al., New England J. of Medicine 339, p. 1734-1738,
1998
[0104] Pertl et al, Semin Perinatol 1999 October;23(5):393-402
[0105] Philip et al, Prenat Diagn 1994 December;14(13):1203-15
[0106] Roche Molecular Biochemicals, Cat. No. 1 796 828
[0107] Samura et al, Prenat Diagn 2000 April;20(4):281-6
[0108] Sekizawa et al, Fetal Diagn Ther 1999
July-August;14(4):229-33
[0109] UNG, Roche Molecular Biochemicals
[0110] U.S. Pat. No. 4,683,102 (Mullis et al.)
[0111] U.S. Pat. No. 4,683,195
[0112] Wang et al, Cytometry 2000 Mar. 1;39(3):224-30
[0113] Whittle, Arch Dis Child 1992 January;67(1 Spec No):65-8
[0114] Wittwer et al., Biotechniques, Vol. 22, No, 1, 130-138,
1997
[0115] WO 97/46707
[0116] WO 97/46712
[0117] WO 97/46714 (Wittwer et al.)
Sequence CWU 1
1
16 1 20 DNA Homo sapiens 1 caccacccag gcatcattag 20 2 23 DNA Homo
sapiens 2 agttcggcag atttcagttc att 23 3 21 DNA Homo sapiens 3
ccacttgccc ctgttaccta a 21 4 22 DNA Homo sapiens 4 gttctcattt
aactgttggc cg 22 5 23 DNA Homo sapiens 5 ggcggatccc ttggcaagtt tac
23 6 24 DNA Homo sapiens 6 cccaaagcag ctctctcgtg tctg 24 7 23 DNA
Homo sapiens 7 ccgtagtagg gtcaggccac ggc 23 8 23 DNA Homo sapiens 8
ccctaatgcc tgcggagttg tgc 23 9 20 DNA Homo sapiens 9 gcctgcattt
gtacgtgaga 20 10 21 DNA Homo sapiens 10 caaagagtgg cagagaaagg a 21
11 23 DNA Homo sapiens 11 gcaggcactg gagtcagaga aaa 23 12 24 DNA
Homo sapiens 12 tgacagcaaa gtctccaatg ttcg 24 13 22 DNA Homo
sapiens 13 tcctccaagc agacttacat cc 22 14 20 DNA Homo sapiens 14
tgggtgcagt ggaactgtca 20 15 24 DNA Homo sapiens 15 caggcagctc
accgagatca cttt 24 16 23 DNA Homo sapiens 16 ggacatggct ttaccttgcg
tcc 23
* * * * *